A variety of tests, both on higher plants and on microalgae, are known for assaying photosynthesis-inhibitory activity. The known measuring principles are based, inter alia, on the fluorescence of chlorophyll or on measuring the photosynthetic oxygen production (B. Hock, C. Fedtke, R. R. Schmidt, Herbizide [Herbicides], Georg Thieme Verlag, 1995, 54 and 112-114; D. Merz, M. Geyer, D. A. Moss, H.-J. Ache, Fresenius J. Anal. Chem, 1996, 354: 299-305). All of these methods, which represent the prior art, have limitations which do not permit high-throughput measurements as they are carried out in the screening of active ingredients, miniaturization, for example a high degree of parallerlization, or a direct couplings with analytical separation techniques for detecting activities in substance mixtures.
Measuring the fluorescence of chlorophyll is an established standard method for studying the photosynthesis process. The methods used in this context rely on fluorimeters which, owing to their methodology, which is based on measurements using probes or cuvettes, only permit serial measurements and are therefore not suitable for high-throughput applications. Moreover, such methods are also very difficult to miniaturize. Typical instruments for this technique are available from the manufacturers mentioned hereinbelow, among others: ADO BioScientific Ltd., Hansatech Instruments, Heinz WaIz GmBH, Qubit Systems Inc.
In the DF-Algentest [DF algae test], water samples are treated with green algae and subsequently measured luminometrically (Methoden der biologischen Wasser-untersuchung [Methods of Biological Water Analytics], Volume 2: Biologische Gewässeruntersuchung, G. Fischer Verlag, 1999, page 386-388). In this test, the first step is the determination of deactivation kinetics for the lagging luminescence of the photosynthesis pigment complex. Conclusions regarding the presence of photosynthesis-inhibitory substances are drawn by comparison with the corresponding deactivation kinetics for an untreated reference sample. This method is only capable of processing samples in series and is thus not suitable for high-throughput measurements.
A further limitation relates to the sample volume for the DF-Algentest, which, owing to the dimensions of the equipment, is in the milliliter order. This method does not allow miniaturization. Moreover, substance mixtures, as are typical for realistic samples, can only be assessed in their entirety by this method. Owing to the possibility of interactions between the sample constituents, there is a risk of false positives.
Tests on higher plants are also known (see, for example, W. Bilger, U. Schreiber, M. Bock, Oecologia 102, 1995, pp. 425-432). These tests provide findings on photosynthetic inhibition via a method in which the fluorescence is measured. Again, the geometry of the test device prevents a high degree of parallerlization and miniaturization. Again, substance mixtures can only be assessed in their entirety.
EP 588 139 A1 describes a test for substance mixtures. The biological effect of the substances in a substance mixture is tested by a combination of chromatographic separation of the substance mixture into the substances to be tested in chromatographic zones, followed by a bioassay (toxicity) of the individual fractions which have been separated. In the bioassay, the individual fractions are brought into contact with luminescence microorganisms which indicate the biological effect of this fraction by means of a local change in their bioluminescence at the individual fractions.
The possibility of parallerlization and miniaturization of activity assays is described in EP 1 043 582 A2. According to the method disclosed in EP 1 043 582 A2, a sensor layer consisting of a diffusion-controlling matrix in which activity sensors are suspended is employed. The bioactivity of the test substances is indicated by optical signals upon contact of this sensor layer with samples.
The object of the invention consists in providing a device and a method for detecting photosynthesis-inhibitory substances which makes possible miniaturization and a markedly higher sample throughput in comparison with the prior art.
The object of the invention is achieved by a method for detecting the photosynthesis-inhibitory activity of substances, comprising the following steps:                providing cells or cell parts with an intact photosystem,        introducing the cells or cell parts into a planar layer,        applying the test substance to the planar layer or into the planar layer,        exciting the luminescence of the cells or cell parts in the planar layer by an excitation light source,        measuring the luminescence of the cells or cell parts in the planar layer by means of a detector, and        associating the detector signal with the degree of photosynthesis inhibition.        